This article was submitted to Biomedical Nanotechnology, a section of the journal Frontiers in Nanotechnology
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Despite extensive efforts to repurpose approved drugs, discover new small molecules, and develop vaccines, COVID-19 pandemic is still claiming victims around the world. The current arsenal of antiviral compounds did not perform well in the past viral infections (e.g., SARS), which casts a shadow of doubt for use against the new SARS-CoV-2. Vaccines should offer the ultimate protection; however, there is limited information about the longevity of the generated immunity and the protection against possible mutations. This study uses Human Coronavirus 229E as a model coronavirus to test the hypothesis that effective delivery of virus-specific siRNAs to infected cells will result in lower viral load and reduced cell death. Two different categories of nucleic acid delivery systems, Peptide/Lipid-Associated Nucleic Acids (PLANAs) and lipophilic polymers, were investigated for their toxicity in human lung fibroblast cells and their ability to deliver specific siRNAs targeting Spike and Envelope proteins in order to prevent cell death in infected cells. Selected siRNAs were effectively delivered to human lung fibroblast cells with negligible toxicity. Cell death due to viral infection was significantly reduced with individual and combinatorial silencing of selected viral proteins. The combinatorial silencing of Spike and Envelope proteins restored the cell viability completely and eliminated plaques in the investigated system. Our cell culture data indicate promising results for the RNAi based approach as an alternative antiviral treatment.
COVID-19 is an emerging disease with little history of therapeutic development. The previous experience with other viral epidemics such as SARS and MERS have revealed difficulties associated with the treatment of similar viral infections. Based on our analysis of the current activity, the researchers are pursuing the two main approaches to manage the current epidemic. On one hand, small molecular drugs are being extensively explored to repurpose them for the treatment of SARS-CoV-2. It is hoped that certain pre-approved drugs will prevent the replication of SARS-CoV-2 and will be widely available since they are approved for human use. It is likely that effective drug hits will emerge from this activity [in one of the first published studies, 29 FDA-approved drugs were identified as “hits” based on their protein interactions maps (
Using RNAi approaches as an antiviral strategy is not unprecedented. Past experiences with SARS have indicated the efficiency of siRNA therapy as a potential antiviral approach. In 2005, Wu et al. reported that siRNAs targeting spike protein (S-protein) of SARS-CoV cause 85–90% reduction in viral load as assessed by PCR analysis (
Developing RNAi-based drugs for SARS-CoV-2 is likely to offer more specific therapies and can potentially be directed against two different categories of targets: 1) viral proteins essential in survival and replication of SARS-CoV-2, and 2) host factors involved in cellular entry and trafficking of the virus. We recently reviewed these RNAi-based strategies and their prospect in COVID-19 treatment in (
Lipofectamine™ 2000 was purchased from Life Technologies (Grand Island, NY, United States). 1,2-Dioleoyl-
All Stars negative control siRNA labeled with Alexa Fluor 488 (AF488; Catalogue number 1027292; sequence: proprietary) was purchased from Qiagen (Valencia, CA, United States). The siRNAs targeting spike (S) and envelope (E) proteins were designed based on the viral RNA sequence reported in literature ( Spike protein: Forward – 5′-GUU AAA UUU GGC AGU GUA UGU UUU UCG-3′ Reverse – 5′-CGA AAA ACA UAC ACU GCC AAA UUU AAC-3′ Envelope protein: Froward – 5′GUU AAA UUU GGC AGU GUA UGU UUU UCG-3′ Reverse – 5′CGA AAA ACA UAC ACU GCC AAA UUU AAC-3′
The polymeric carrier used in this study was LeuFect B (batch numbers 12-18-6A and 12-18-9A) from RJH Biosciences (Edmonton, Canada). The components and preparation methods for PLANAs have been previously reported (
Human MRC-5 lung fibroblast cells (ATCC® CCL-171™) were thawed and subcultured using Dulbecco’s Modified Eagle Medium (DMEM) low glucose. Cells were incubated in 37°C and 5% CO2 for the entire growth time. Cells were expanded when reached ∼80% confluency and were replaced after 30 expansions or 90 days (whichever earlier).
Human Coronavirus 229E was obtained from ATCC (VR-740). The commercial stock was used to infect MRC-5 cells and initially amplified via two rounds of supernatant transfer (p1 and p2). 50 ml of p2 supernatant was retained as a seed stock. Final virus stock for experiments was produced by infecting 5x T-185 flasks of MRC-5 cells with 1 ml each of p2 seed stock in DMEM+2% FBS. Infection was allowed to proceed for 5 days until 90% of MRC-5 cells showed cytopathic effect. Culture supernatant was collected, and cell material was pelleted via centrifugation at 1000 ×g for 15 min. The clarified supernatant was kept on ice. Fresh serum-free DMEM was added to the culture vessels, and remaining cells were harvested using a cell scraper and pelleted as above. The two cell pellets were combined in a total of 5 ml of serum-free DMEM and subjected to three rapid freeze-thaw cycles. Cell lysates were added back to the clarified culture supernatant, and 1 ml aliquots of virus preparation were stored at -80 C for experiments. p3 stock virus was quantitated via TCID50 method on MRC5 cells using the MTT assay described below.
MRC-5 cells were seeded in 96-well plates at ∼50% confluency 24 h before the experiment. The study groups included: No Treatment (NT; normal saline) and scrambled siRNA delivered by Leu-Fect B (12-18-6A and 12-18-9A), Lipofectamine™ 2000, and PLANAs. The siRNA/Lipofectamine™ complexes and siRNA/polymer polyplexes were prepared according to the manufacturers’ guidelines. Briefly, Lipofectamine™ and siRNA were diluted in FBS- and antibiotic-free OPTIMEM and were mixed. After 20 min. incubation at ambient temperature, the cell culture medium was removed, and the siRNA complexes were added to the wells in OPTIMEM. After 6 h of incubation at 37°C, the complexes were removed, and the cell culture medium was added to the wells. For polyplexes, polymer and siRNA were mixed in normal saline, and after 30 min of incubation in ambient temperature, the polyplexes were added to the cells. For each carrier, siRNA was delivered at two final concentrations (in triplicates): 50 and 100 nM, which also showed exposure to two different carrier concentrations. Cells were incubated in 37 C and 5% CO2 for 48 h after exposure. A Cell Counting 8 (CCK8) KIT (also known as WST-8) was used to evaluate the potential toxicity of the formulations on the viability of the MRC-5 cells. After the 24 h incubation period, 10 uL of CCK solution (Cat. #B34304) was added to each well. The plates were incubated at 37 C for 1 h, after which the absorbance was measured at 450 nm using a SpectraMax M5 UV VIZ Plate Reader to determine the percentage of viable cells compared to the no treatment group (after eliminating the signal from “Blank” wells containing cell-less medium with CCK-8 solution added).
MRC-5 cells were seeded in 24-well plates at ∼50% confluency the day before the experiment and were treated with one of the following study groups: Normal saline (no treatment; NT), free AF488-siRNA, AF488-siRNA/polymer complexes, PLANA encapsulated AF488-siRNA, or Lipofectamine™ 2000 encapsulating AF488-siRNA (all in triplicates). The final concentration of delivered siRNA was 36 nM for all treatment groups. After exposure to siRNA, cells were incubated at 37°C and 5% CO2 for 24 h, and then the media was removed, and the cells were detached using 0.05% trypsin (for Lipofectamine the manufacturer’s protocol was followed). Trypsinized cells were fixed using 3.7% formaldehyde in 1X PBS, and each sample was evaluated using FACSVERSE flow cytometer (BD Biosciences; San Jose, CA). The fluorescein isothiocyanate (FITC) channel was used to quantify cell-associated fluorescence. The percentage of cells positive for fluorescence signal and the mean fluorescence of the cell population were calculated following each flow cytometry analysis using the calibration of the signal gated with No Treatment cells in order to eliminate autofluorescence of approximately 1% of the population in “no treatment” group.
A sterile cover lip was placed at the bottom of each well in 6-well plates, and the cover slips were covered with a 10% FBS solution in DMEM and were incubated for 30 min at 37 C to enhance the cell adherence to the cover slip surface. MRC-5 cells were then seeded in the wells at a confluency of ∼70% and were incubated overnight at 37 C and 5% CO2. Cells were then treated with one of the following groups: free AF488-siRNA, Lipofectamine™ 2000 encapsulating AF488-siRNA, AF488-siRNA/polymer complexes, or PLANA encapsulated AF488-siRNA (all in triplicates). Cells were then incubated for 24 at 37 C and 5% CO2, after which the media was removed, and cells were washed three times with one X PBS. Cells were then fixed with 3.7% formaldehyde in 1X PBS for 10 min. The fixed cells were rinsed three times in 1X PBS for 5 min, after which Texas Red Phalloidin solution (40 uL and 10 mg BSA in 10 ml of 1X PBS) was added to the cells to stain the cell membrane. Stained cells were incubated at room temperature for 1 h, and were then washed three times with 1X PBS for 5 min. One drop of VECTASHIELD VIBRANCE with DAPI was added to each slide to stain the nucleus, and the coverslips were placed face down on slides, without air bubbles, and were stored overnight away from light to dry. Once dry, a Nikon A1R high-definition resonant scanning confocal microscope and a NIS-Elements software (AR 4.30.02, 64bit) were used to image the cells.
The antiviral studies were performed to test the efficacy of targeting spike and envelope proteins individually and simultaneously, to investigate the potential benefit of combinatorial silencing of two viral targets via delivering a cocktail of siRNAs. For this set of studies, remdesivir was used as a positive control, and media only was used as a negative control. Polymer/siRNA complexes or PLANAs were prepared to deliver 100 nM of siRNA targeting mRNA sequence for spike protein expression, 100 nM of siRNA targeting mRNA sequence for envelope protein, or a combination of 50 nM of each siRNA. Treatments were added to the plate at 1 h prior to infection. P3 virus stock was added at 2 TCID50 units per well in a total of 100 µL of serum-free DMEM and incubated at 37 C for one hour. After the initial incubation, an additional 100 µL of DMEM containing 4% FBS was added to each well to make a final concentration of 2% FBS and incubated at 37 C, 5% CO2. At 7 days post-infection, cell viability was assessed using MTT assay (Promega) according to manufacturer instructions. Briefly, media was removed and discarded to leave 100 µL in each experimental well. MTT reagents were mixed and added directly to cultures (20 µL/well) and allowed to incubate for 1 h at 37 C. 50µL of 10% SDS was then added to stop the MTT reaction and inactivate the virus for analysis. Inactivation was allowed to proceed for 18 h at room temperature. After inactivation, the MTT colorimetric signal was analyzed using a Spectramax M5 plate reader.
MRC-5 cells were seeded in 24-well plates at 1 × 105 cells per well 24 h before the experiment. Polymer/siRNA complexes or PLANAs were prepared to deliver 100 nM of siRNA targeting mRNA sequence for spike protein expression, 100 nM of siRNA targeting mRNA sequence for envelope protein, or a combination of 50 nM of each siRNA and were added to the wells one hour prior to infection. 5-fold dilutions of p3 229E virus stock in 500 µL of serum-free DMEM were then added in triplicate such that, for each treatment condition, three wells were infected at TCID50 8, 1.6, 0.32, and Mock (no virus). After one hour of infection at 37 C, overlay media containing 1% Agarose in DMEM was heated to 50 C. Immediately prior to overlay, 4% FBS was added to the overlay media, 500 μL of overlay was added to each well, and plates were cultured for 4 days at 37 C, 5% CO2. At 4 dpi, wells were fixed by adding 1 ml of 10% formaldehyde and incubating at 37 C overnight. After fixation, media was removed, and monolayers were stained with 1% crystal violet in 20% ethanol for 15 min at room temperature, followed by several rinses with diH2O and drying before plaques were counted.
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The toxicity of the siRNA delivery system plays a crucial role in the safety profile of this approach in a clinical setting. Since the proposed approach could potentially be used via the inhalation route, especially in the early stages of the SARS-CoV-2 infection, we selected MRC-5 human lung fibroblast cells for our toxicity experiments. The cell viability results after 24 h exposure to different carriers are summarized in
The cytotoxicity of the selected siRNA/carriers in human MRC-5 lung fibroblast cells quantified using the CCK assay. No statistically significant difference was observed in the viability of cells exposed to either of the polyplexes or PLANA compared to cells exposed to normal saline (No Treatment, or NT). * indicates that the viability of the cells exposed to 100 nM of siRNA delivered with Lipofectamine™ was significantly lower compared to NT group.
The toxicity of siRNA delivery systems has been a limiting factor for their clinical use as well as extensive applications
Internalization of free siRNA without a carrier into target cells is usually negligible, which is observed in our experiments as well (
The level of cellular internalization of AlexaFluor488-labeled siRNA in human MRC-5 cells using the selected carriers. All Selected delivery systems included in the study created mean fluorescence values comparable to Lipofectamine™ (included as positive control). * indicates statistically significant difference with PLANA group (α = 0.013). ** indicates significant difference with both PLANA (α = 0.002) and LipofectamineTM (α = 0.034) groups. One-way ANOVA was used for statistical analysis.
The cellular internalization was visualized using confocal microscopy and the results were confirmed by flow cytometry (
The confocal microscopy images of AF488-labeled siRNA (green channel) delivered to MRC-5 cells by Lipofectamine and PLANAs as compared to free AF488-labeled siRNA. DAPI (blue channel) and Texas Red (Red channel) were used to dye the nuclei and cell membranes, respectively.
We assessed the viability of viral transduced cells and plaque formation to determine the effect of siRNA delivery in inhibiting viral infection on MRC-5 cells. The viability of healthy and hCoV-229E-infected MRC-5 cells was evaluated using the MTT assay after siRNA delivery targeting S-protein, E-protein, or a combination of both (each representing half of the concentration compared to individual siRNA groups) in a variety of concentrations (ranging from 25 to 100 nM of total siRNA). The untreated MRC-5 cells were included to confirm the safety profile observed in cytotoxicity studies when siRNAs were delivered to the cells. Remdesivir was used as the positive control in this set of studies.
The siRNA delivery (in the absence of viral infection) did not affect the cell viability significantly, which again confirmed the minimal cytotoxicity of the siRNA treatments on the lung cells. The lowest viability observed in the normal MRC-5 cells was in the cells exposed to E-protein siRNA lipopolyplex (∼81 and ∼89% viability for 100 nM of siRNA delivered by 1.2-18-6A and 1.2-18-9A, respectively:
Cell viability of healthy (no virus or NV) and hCoV-229E-infected (Virus) MRC-5 cells after delivering 25, 50, and 100 nM of siRNA targeting Spike (S), Envelope (E), or a combination of both siRNAs (1:1 ratio) using 12–18-6A
The efficiency of the reported approach should be additionally evaluated by quantifying the viral RNA load in infected cells using real-time PCR. However, we demonstrated effective internalization of siRNA in MRC-5 cells in this manuscript and have previously reported the silencing efficiency of the selected delivery systems in different cell lines after delivering the siRNA cargo to the cytoplasm (
Finally, we used plaque assay to confirm the efficiency of the siRNA approach in preventing plaques formed in MRC-5 monolayers as a result of hCoV-229E-infection. The plaque reduction neutralization test (PRNT) is considered a “gold standard” in the evaluation of antiviral strategies used against SARS-CoV-2 infections (
Targeting S-protein and E-protein via PLANAs completely eliminated plaques seen at 8 TCID50 units per well in no treatment group (black arrows). Experiments were conducted in triplicates.
The deployment of the siRNA as an alternative approach to upper respiratory tract viral infections is not unprecedented. Many manuscripts reviewed the possibility of using RNA interference (RNAi) as a therapeutic approach against COVID-19 (
In conclusion, we showed the efficiency of an siRNA-based anti-viral approach
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The study design was a collaboration among HMA, HU, and KP. The experimental methods were mostly performed by PM. The viral infection experiments were designed and performed by JT. Data analysis was performed as a collaboration among HMA, HU, and KP. Manuscript was written by HMA, and was revised and modified by HU and KP.
This study was partially supported by a Research Starter Grant in Pharmaceutics from PhRMA Foundation. The authors would also like to thank Chapman University School of Pharmacy for the financial support provided to graduate students and Faculty. Additional funding was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC; Uludag Lab).
Author HU is a shareholder in RJH Biosciences Inc. which had the following involvement with the study: RJH Biosciences Inc. provided materials for this study.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Remant K.C. for the preparation of the Leu-Fect B materials used in this study. HU is a shareholder in RJH Biosciences Inc., which provided materials for this study, and declare the financial conflict of interests due to ownership in RJH Biosciences Inc.
The Supplementary Material for this article can be found online at: